The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system uses a 19-20 nucleotide (nt) guide RNA (gRNA) to direct the Cas9 endonuclease to introduce a DNA double-strand (ds) break (DSB) at practically any specified site in the genome. The DSB is usually repaired by non-homologous end joining (NHEJ), resulting in a small insertion or deletion (indel) that disrupts the target gene. If a homologous donor DNA is introduced into cells at the same time, the DSB can also be repaired by homology-dependent repair (HDR), leading to gene editing. The power of the CRISPR technology has made it possible to disrupt all of the predicted genes in the human genome, revealing that there are approximately 2000 core essential genes and many other context-dependent genes.
Despite the success of CRISPR, the efficiency is still highly variable due to multiple factors, such as guide RNA effectiveness, transfection efficiency, retrovirus titer, and cell type. This problem is compounded exponentially when one needs to target sequentially two or more genes (iterative gene targeting) to analyze their functional relationship. Various methods have thus been developed to enrich cells that have been successfully targeted. A common enrichment strategy is to use markers, such GFP, drug resistance, or death receptors to select for cells that have been transfected or infected. However, the expression of these markers does not necessarily correlate with high levels of Cas9 or gRNAs. Selection for drug resistance also leads to the integration of foreign DNA into the chromosome, which is itself an alteration to the genome and risks oncogenic transformation. For iterative gene targeting, if each round uses a separate drug resistance gene, one would quickly run out of choice and the genome would be littered with foreign DNA.
One alternative enrichment method uses co-targeting of the HPRT gene. This gene encodes an enzyme that catalyzes the conversion of hypoxanthine to inosinemonophosphate and guanine to guanosine monophosphate in the non-essential purine salvage pathway. HPRT-positive cells are sensitive to 6-TG, which is converted to the cytotoxic nucleotide form by HPRT. The strategy is to co-express Cas9 with two guide RNAs, one against HPRT and the other against the gene of interest. The resulting 6-TG resistant cells are highly enriched for mutations in the gene of interest with no other genetic imprints.
One unique feature of the HPRT gene is that it can be both selected and counterselected. HPRT mutant cells are resistant to 6-TG but sensitive to HAT. In HAT media, the de novo synthesis of nucleotides is blocked by aminopterin, a strong inhibitor of the key enzyme dihydrofolate reductase (DHFR). Cells have to rely on the exogenously provided hypoxanthine and thymidine to synthesize nucleotides via the HPRT-dependent salvage pathway. As such, only HPRT wild type cells can survive and proliferate after HAT selection.